Robotic method for coating a multiwell plate by a polyelectrolyte multilayer film

ABSTRACT

The invention concerns a robotic method for coating the bottom surface of at least one well of a multiwell plate by a polyelectrolyte multilayer film, the multiwell plate obtainable according to the method and the use thereof for cell culture.

This application is a division of U.S. application Ser. No. 16/468,806,filed Jun. 12, 2019, which is the U.S. National Stage of InternationalApplication No. PCT/EP2017/083092, filed Dec. 15, 2017, which claims thepriority of European Patent Application No. 16306697.0, filed Dec. 15,2016, all of which are hereby incorporated by reference.

The present invention concerns a robotic method for coating a multiwellplate by a polyelectrolyte multilayer film allowing the preparation offilms in a highly reproducible and reliable manner and with a very highspatial homogeneity.

The deposition of polyelectrolyte multilayer (PEM) has emerged as a veryeasy handling and versatile tool. Based on the alternate adsorption ofpolycations and polyanions, this technique allows to buildup films withtunable properties: by adjusting several parameters such as the chemicalnature of the polyelectrolytes, pH and ionic strength, immersion andrinsing times, post-treatment of the film, it is possible to obtain analmost infinite variety of architectures.

The electrostatic layer-by-layer (LbL) assembly technique has recentlyemerged as a very promising tool for biomedical applications in view ofits versatility, notably the large range of building blocks and assemblyconditions, and the possibility to deliver locally small therapeuticsand proteins, such as growth factors. The large range of templates(shape, size), of layer materials (biologic or synthetic) available forsurface modification and the impressive panel of LbL assemblytechnologies have all contributed to the widespread use of thetechnique.

Application EP 1 535 952 discloses a method for preparing cross-linkedpolyelectrolyte multilayer films, comprising the reaction ofcomplementary functional groups: carboxylic groups and amino groups,present in the polymers that constitute the multilayer film, in thepresence of a coupling agent, whereby amide bonds are formed.

Application WO 2010/081884 discloses a method for coating a surface,comprising the following steps: (a) sequentially depositing on a surfaceat least one layer of alternate adsorbed polyelectrolytes to provide acoated surface presenting complementary amino and carboxylic reactivegroups, wherein a first (or conversely second) polyelectrolyte is acationic polymer comprising said amino groups and a second (orconversely first) polyelectrolyte is an anionic polymer comprising saidcarboxylic groups, (b) reacting said complementary reactive groups ofthe coated surface in the presence of a coupling agent, as to form amidebonds between said complementary reactive groups giving rise to across-linked polyelectrolyte multilayer film, and (c) treating saidcross-linked polyelectrolyte multilayer film with a protein containingsolution, preferably with a growth factor type protein containingsolution, as to incorporate said protein on and inside said cross-linkedpolyelectrolyte multilayer film. The obtained film thus containsproteins. Films were constructed in 96 well plates, but manually using amultiple channel pipette for dispense and aspiration steps. However,this process is user-dependent and thus not reproducible, time-consumingand susceptible to human errors.

A variety of strategies have already been developed to automate LbLfilms deposition. While the most popular techniques are dip coating,spraying and spin coating, these technologies are rather suited forlarge substrates, require a significant volume of liquid and cannot beadapted to cell culture plates.

Recently, Jaklenec et al. (ACS Appl Mater Interfaces, 2016,8(3):2255-61) used an automated liquid handling robot, to preparepolyelectrolyte films from synthetic polymers (polyacrylicacid/polyallylamine hydrochloride, PAA/PAH) over a large pH range andcontaining increasing number of layers. To this end, the polyelectrolytefilms were built on (bottomless) silicon 96 well plates glued to siliconwafer, in order to be subsequently analyzed by profilometry and used toscreen for cell attachment and spreading. However, the authorsidentified several remaining technical problems: i) radial heterogeneityin film thickness, in particular coffee ring effect; ii) difficulty toautomate characterization and analysis of the film. Also, there wasneither characterization of the polyelectrolyte films in situ in thecell culture plates, nor proofs that bioactive molecules can be loadedin films and be effectively bioactive.

Accordingly, the development of an automated electrostaticlayer-by-layer assembly technique in multiple-well plates allowing thepreparation of films:

-   -   in a highly reproducible and reliable manner,    -   with a very high spatial homogeneity inside each well and        between different wells in order to optimize cell culture,        is still required.

For this purpose, the present invention concerns a method for coatingthe bottom surface of at least one well of a multiwell plate by apolyelectrolyte multilayer film, said method comprising n successivesequences, n being an integer from 1 to 2000, wherein each sequencecomprises the steps of:

-   a) robotic deposit of a volume V_(PE) ¹ of a solution of a first    polyelectrolyte PE¹ on the bottom surface of at least one well of a    multiwell plate, wherein the first polyelectrolyte PE¹ is either a    cationic polymer comprising amino groups, or an anionic polymer,    then-   b) robotic aspiration of an aspirated volume V_(aspPE) ¹ of said    solution of PE¹, wherein the aspirated volume V_(aspPE) ¹ is higher    than or equal to V_(PE) ¹, then-   c) robotic deposit of a volume V_(PE) ² of a solution of a second    polyelectrolyte PE² on said bottom surface, wherein the second    polyelectrolyte PE² is a cationic polymer comprising amino groups    when PE¹ is an anionic polymer, or PE² is an anionic polymer when    PE¹ is a cationic polymer comprising amino groups, then-   d) robotic aspiration of an aspirated volume V_(aspPE) ² of said    solution of PE², wherein the aspirated volume V_(aspPE) ² is higher    than or equal to V_(PE) ².

The robotic steps are typically carried out with an automated liquidhandling machine provided with pipetting arm as robot, for example witha TECAN Freedom EVO® 100 robot. The ends of the arms of the robots areprovided with tips which allow depositing and aspirating solutions. Theliquid handling arm pipettes the solutions in their respectivereservoirs and dispensed them in selected wells. After being aspirated,the solutions are thrown in a trash.

Steps a) and c) are dispense steps. At step a), the robot aspirates thesolution of the first polyelectrolyte PE¹ from a reservoir containingsaid solution and transfers said solution to the bottom surface of atleast one well of the multiwell plate. At step c), the robot aspiratesthe solution of the second polyelectrolyte PE² from a reservoircontaining said solution and transfers said solution to the bottomsurface of the well(s) of the multiwell plate. Preferably, to implementsteps a) and c), the ends of the arms of the robots are provided with 1mL pipette tips.

Steps b) and d) are aspiration steps. At step b) and d), the robotaspirates the solution from the well and transfers it to the trash.Preferably, to implement steps b) and d), the ends of the arms of therobots are provided with 200 μL pipette tips. These smaller tips allow abetter accuracy of the aspiration steps and thus a better homogeneity ofthe obtained film.

The typical pipetting speed is from 400 to 800 μL/s for dispense stepsa) and c) and from 30 to 150 μL/s for aspiration steps b) and d).

Preferably, each sequence of the method comprises:

-   -   between steps a) and b), a step a′) of incubation wherein the        solution of first polyelectrolyte PE¹ is left in contact with        the bottom surface for a duration from 1 to 30 minutes,        preferably from 5 to 15 minutes, and    -   between steps c) and d), a step c′) of incubation wherein the        solution of second polyelectrolyte PE² is left in contact with        the bottom surface for a duration from 1 to 30 minutes,        preferably from 5 to 15 minutes.

The method comprises steps b) and d) of robotic aspiration of anaspirated volume V_(aspPE) of the solution of PE, wherein the aspiratedvolume V_(aspPE) is higher than or equal to V_(PE), PE being either PE′step b) or PE² (step d)).

In one embodiment, V_(aspPE) ¹=V_(PE) ¹ and V_(aspPE) ²=V_(PE) ².

In another embodiment, V_(aspPE) ¹ is higher than V_(PE) ¹ and V_(aspPE)² is higher than V_(PE) ². Typically, V_(aspPE) ¹ and V_(aspPE) ² arerespectively higher than 1.01×V_(aspPE) ¹ and 1.01×V_(aspPE) ², notablyhigher than 1.02×V_(aspPE) ¹ and 1.02×V_(aspPE) ², for example higherthan 1.05×V_(aspPE) ¹ and 1.05×V_(aspPE) ², most preferably around1.10×V_(aspPE) ². Generally, V_(aspPE) ¹ and V_(aspPE) ² arerespectively lower than 1.50×V_(aspPE) ¹ and 1.50×V_(aspPE) ².

This means that the arm of the robot aspirates an additional aspirationvolume compared to the volume of solution which has been deposited. Infact, the robot aspirates a mixture of solution which has been depositedand of air.

The present invention is based on the discovery that aspirating a volumehigher than or equal to the volume of solution which was deposited leadsto a film having a high spatial homogeneity, i.e. not only a highspatial homogeneity of the film deposited inside each individual well,but also to a high reproducibility of the film deposit between differentwells (within a single plate and in different plates built inindependent runs of the automated machine).

Using a robotic arm equipped with multiple channels enables to automatethe layer-by-layer deposit method and to optimize the workflow. Parallelprocessing provides several key advantages for the LbL film buildup: i)ensuring the reproducibility of the deposit, ii) enhancing thethroughput for cell screening assays, iii) being adaptable to any typeof robotic arm whatever the manufacturer.

The implementation of the method according to the invention oncommercially available liquid handling machines will undoubtedly broadenthe range of possibilities offered by the well-controlled LbL coatingsin view of applications in biomolecular or cellular screening forbiotechnologies and regenerative medicine.

The method involves a multiwell plate, for example 6-, 12-, 24-, 48- or96-well plates. The 96-well plate format is particularly interesting inview of cell screening assays, since multiple conditions can be screenedin parallel and the number of cells needed per well is small (typically5000 cells/well or less).

It is possible to implement the method on all wells of the multiwellplate, or only on some of them (preferably at least on two wells).

The wells chosen for the deposit at step a) of the first sequence arethe wells of the multiwell plate on which the multilayer film is formed.Generally, all the steps of the method are carried out on the samewells, m being the number of wells selected by the used to implement themethod. Accordingly, m is an integer from 1 (embodiment wherein themethod is implemented in only one well) to the number of wells of themultiwell plate (for example 96 for a 96-well plate), preferably from 2to the number of wells of the multiwell plate. Indeed, in order to buildthe layer pairs of the multilayer film, the deposit or aspirations ofsteps b), c) and d) are carried out on the same wells than the wells onthe bottom surface of which the solution of the first polyelectrolytePE¹ is deposited at step a) of the first sequence. Similarly, all thesteps of all the sequences involve the same wells than the one chosen atstep a) of the first sequence. The software of the automated handlingrobot allows defining the number of wells m and specific positions wherethe layer-by-layer film must to be deposited.

All the wells of the plate have a height H. The bottom of the wells mayhave various forms, U form, V form or planar round from. The wellspreferably have a planar bottom, usually a round bottom surface having adiameter d.

Each sequence of the method allows depositing a layer pair of themultilayer film. The multilayer film obtained at the end of the methodcomprises n layer pairs.

The number “n” of layer pairs in polyelectrolyte multilayer filmprepared through the method can vary in a wide range and depend on thedesired thickness. Preferably, n is an integer from 2 to 2000, inparticular from 5 to 2000, preferably from 5 to 1000, more preferablyfrom 5 to 100. When a thick polyelectrolyte film is desired, n can varyfrom 15 to 1000, preferably from 20 to 500 (in particular from 20 to60).

Each layer pair comprises a layer of polyelectrolyte PE¹ and a layer ofpolyelectrolyte PE² of opposite charge. The film architecture isprecisely designed and can be controlled to 1 nm precision with a rangefrom 1 to 50 000 nm, preferably from 100 nm to 30 μm. The thickness ofthe film can generally vary from 1 nm to 50 000 nm, preferably from 500nm to 20 μm, more preferably from 1 to 10 μm. A film is considered as athick film when its thickness is more than 300 nm.

The method comprise depositing sequentially a first polyelectrolyte PE¹,then a second polyelectrolyte PE², one of them being a cationic polymercomprising amino groups, and the other being an anionic polymer.

In the present invention, the terms “anionic polymer” relate to apolymer comprising at least one group susceptible to bear a negativecharge. The anionic polymer preferably comprises carboxylic, phosphate,sulfate and/or sulfonate groups. The carboxylic groups can be present inthe form of acids, acid halide (preferably, acid chloride), acidanhydride or activated esters, such as N-hydroxysulfosuccinimide esteror n-paranitrophenyl ester. The anionic groups are covalently bond tothe used polymers.

Any anionic polymer can be used in the method, including, withoutlimitation thereto, poly(acrylic) acid, poly(methacrylic) acid,poly(glutamic) acid, polyuronic acid (alginic, galacturonic, glucuronic,. . . ), glycosaminoglycans (hyaluronic acid or a salt thereof (such assodium)—also called hyaluronan-, dermatan sulphate, chondroitinsulphate, heparin, heparan sulphate, keratan sulphate), poly(asparticacid) and Polystyrene sulfonate (PSS), any combination of thepolyamino-acids (in the D and/or L forms), and mixtures thereof.

The terms “cationic polymer” relate to a polymer comprising at least onegroup susceptible to bear a positive charge.

In the sense of the invention, the amino groups can be present in theform of hydroxylamine, hydrazide and amine functions. The amino groupsare covalently bond to the used polymers.

Any cationic polymer comprising amino groups can be used in the method,including, without limitation thereto, poly(lysine), such as poly(D-,L-lysine), poly(diallydimethylammonium chloride), poly(allylamine),poly(ethylene)imine, chitosan, polyarginine, such as Poly(L-arginine),Poly(ornithine), polyhistidine, such as Poly(D,L-histidine),poly(mannosamine), polyallylamine hydrochloride (PAH) and more generallyany combination of the polyamino acids (in the D and/or L forms), andmixtures thereof.

Preferably, the cationic polymer comprising amino group ispoly(L-lysine) (or PLL), polyallylamine hydrochloride (PAH) or chitosan(CHI) and/or the anionic polymer comprising amino group is thehyaluronic acid or a salt hereof, such as hyaluronan sodium (also calledgenerally HA), heparin (HEP), polystyrene sulfonate (PSS),poly(L-glutamic acid) (PGA) or a mixture thereof.

The polyelectrolyte multilayer film is more preferably a (PLL/HA) film,a (PSS/PAH) film, a (PLL/PGA) or a (CHI/PGA) film.

The molecular weight of the polymers identified above can vary in a widerange. More preferably, the molecular weight is in the range from 0.5kDa to 20,000 kDa, even more preferably, the molecular weight is in therange from 5 to 2,000 kDa.

Generally, the same polyelectrolytes PE¹ and PE² are used for all thesequences. Generally, the same solution of first polyelectrolyte PE¹ isused for all steps a) of each sequence, and the same solution of secondpolyelectrolyte PE² is used for all steps c) of each sequence.

Generally, V_(PE) ¹ is the same for all the sequences, V_(aspPE) ¹ isthe same for all the sequences, V_(PE) ² is the same for all thesequences and V_(aspPE) ² is the same for all the sequences.

Generally, the method comprises at least a rinsing step after eachpolyelectrolyte deposit step. Accordingly, the method usually comprises:

-   -   between steps b) and c) of each sequence, a rinsing step        comprising the following substeps:    -   b2) robotic deposit of a volume V_(rinsePE) ¹ of a rinsing        solution on said bottom surface, then    -   b3) robotic aspiration of an aspirated volume V_(asprinsePE) ¹        of said rinsing solution, wherein the aspirated volume        V_(asprinsePE) ¹ is higher than or equal to V_(rinsePE) ¹,    -   wherein the rinsing step can be repeated, and    -   after step d) of each sequence, a rinsing step comprising the        following substeps:    -   d2) robotic deposit of a volume V_(rinsePE) ² of a rinsing        solution on said bottom surface, then    -   d3) robotic aspiration of an aspirated volume V_(asprinsePE) ²        of said rinsing solution, wherein the aspirated volume        V_(asprinsePE) ² is higher than or equal to V_(rinsePE) ²,        wherein the rinsing step can be repeated.

At steps b2) and d2, the robot aspirates the rinsing solution from areservoir containing said solution and transfers said solution to thebottom surface of the well(s) of the multiwell plate. Preferably, toimplement steps b2) and d2), the ends of the arms of the robots areprovided with 1 mL pipette tips.

At steps b3) and d3), the robot aspirates the solution from the well andtransfers it to the trash. Preferably, to implement steps b3) and d3),the ends of the arms of the robots are provided with 200 μL pipettetips.

Usually, when V_(aspPE) ¹=V_(PE) ¹ and V_(aspPE) ²=V_(PE) ², thenV_(asprinsePE) ¹=V_(rinsePE) ¹ and V_(asprinsePE) ²=V_(rinsePE) ². Inthe same way, usually, when V_(aspPE) ¹ is higher than V_(PE) ¹ andV_(aspPE) ² is higher than V_(PE) ², then V_(asprinsePE) ¹ is higherthan V_(rinsePE) ¹ and V_(asprinsePE) ² is higher than V_(rinsePE) ².Typically, the same additional volume percentage is used for all ofthem. In other words, typically, V_(aspPE) ¹=β×V_(PE) ¹, V_(aspPE)²=β×V_(PE) ², V_(asprinsePE) ¹=β×V_(rinsePE) ¹ and V_(asprinsePE)²=β×V_(rinsePE) ², wherein β is a number from 1.00 (i.e. no additionalvolume aspirated) to 1.30.

Preferably, when the method comprises these rinsing steps, each sequencethereof also comprises:

-   -   between steps b2) and b3), a step b2′) of incubation wherein the        rinsing solution is left in contact with the bottom surface for        a duration from 0.2 to 10 minutes, preferably from 1 to 5        minutes, and    -   between steps d2) and d3), a step d2′) of incubation wherein the        rinsing solution is left in contact with the bottom surface for        a duration from 0.2 to 10 minutes, preferably from 1 to 5        minutes.

Generally, the same rinsing solution is used for all steps b2′) for allthe sequences and the same rinsing solution is used for all steps d2′)for all the sequences.

If possible, the rinsing solution used at step b2) is the same than theone used at step d2), since this facilitates the method. Generally, whenthe same rinsing solution is used both for steps b2) and d2), the methodinvolves three solutions: the solution of a first polyelectrolyte PE¹,the solution of second polyelectrolyte PE² and the rinsing solution usedboth for steps b2) and d2).

If different rinsing solutions are required at steps b2 and d2), themethod typically involves four solutions: the solution of a firstpolyelectrolyte PE¹, the solution of second polyelectrolyte PE², and therinsing solution used for step b2) and the rinsing solution used forstep d2).

Suitable solvents for polyelectrolyte solutions and rinsing solutionsare: water, aqueous solutions of salts (for example NaCl, KCl, MnCl₂,(NH₄)₂SO₄), any type of physiological buffer (Hepes, phosphate buffer,culture medium such as minimum essential medium, Mes-Tris, Mes, Tris,buffers) and water-miscible, non-ionic solvents, such as C1-C4-alkanols,C3-C6-ketones including cyclohexanone, tetrahydrofuran, dioxane,dimethyl sulphoxide, ethylene glycol, propylene glycol and oligomers ofethylene glycol and propylene glycol and ethers thereof and open-chainand cyclic amides, such as dimethylformamide, dimethylacetamide,N-methylpyrrolidone and others. Polar, water-immiscible solvents, suchas chloroform or methylene chloride, which can contain a portion of theabovementioned organic solvents, insofar as they are miscible with them,will only be considered in special cases. Water or solvent mixtures, onecomponent of which is water, are preferably used. If permitted by thesolubility of the polyelectrolytes implemented, only water is used asthe solvent, since this simplifies the method.

In order to improve further spatial homogeneity of the film formed, twodifferent embodiments have been developed and are described hereafter:

In a first embodiment, called “wet surface” hereafter, a volume V_(wet)of the first polyelectrolyte PE¹ solution is added initially (i.e. priorto buildup of the film, prior to the sequences described above) to eachwell, typically in order to form a liquid layer recovering the bottomsurface.

In this first embodiment, in the method defined above, prior to step a)of the first sequence, the at least one well on which step a) will becarried out is filled with a volume V_(wet) of the solution of the firstpolyelectrolyte PE¹, wherein the volume V_(wet), expressed in mL, isfrom:

V _(wet) ^(min)=0.5×π(d/2)²

to

V _(wet) ^(max)(2/3)×π(d/2)² ×H,

wherein:

-   -   d is the diameter of the well, expressed in mm,    -   H is the height of the well, expressed in mm.

The volume V_(wet) depends on the diameter (d) and height (H) of thewells of the multiwell plate.

The volume V_(wet) is higher than or equal to V_(wet)^(min)=0.5×π(d/2)², which corresponds to a volume allowing a liquidheight of 0.5 mm, which is considered to be the lowest liquid height.

The volume V_(wet) is lower than or equal to V_(wet)^(max)=(2/3)×π(d/2)²×H, which corresponds to a volume allowing a liquidheight of (2/3) of the height of the well. Preferably, the volumeV_(wet) is lower than or equal to (1/2)×π(d/2)²×H, or even lower than orequal to (1/3)×π(d/2)²×H.

Table 1 below provides examples of usual heights and diameters ofmultiwell plates and the corresponding V_(wet) ^(min) and V_(wet)^(max).

TABLE 1 usual H and d of multiwell plates and corresponding V_(wet)^(min) and V_(wet) ^(max) Number of wells 96 wells 48 wells 24 wells 12wells 6 wells Height H (mm)  11.20  16.84  17.80  18.00   17.67 diameterd (mm)  6.40  11.00  15.60  22.10   34.80 Total 360.00 1600.00 3400.006900.00 16800.00 volume/well (μL) V_(wet) ^(min) (μL)  16.1  47.5  95.5 191.7  475.4 V_(wet) ^(max) (μL) 240.0 1066.7 2266.7 4600.0 11200.0

Preferably, the volume V_(wet) is the minimal recommended working volumeof the multiwell plate, i.e. the minimal working volume recommended onthe data sheet of the multiwell plate.

Two alternatives are possible to obtain a well filled with a volumeV_(wet) of a solution of the first polyelectrolyte PE.

According to one alternative, the method comprises, prior to step a) ofthe first sequence, a step a0) of robotic deposit of a volume V_(wet) ofthe solution of the first polyelectrolyte PE′ on the bottom surface ofeach well on which the deposit will be carried out at step a) of thefirst sequence.

This alternative is interesting in that only one additional step isnecessary.

According to a second alternative, the method comprises, prior to stepa) of the first sequence, the steps of:

a1) robotic deposit of a volume V_(a1)) of the solution of the firstpolyelectrolyte PE¹ on the bottom surface of each well on which thedeposit will be carried out at step a) of the first sequence, whereinV_(a1)) is higher than V_(wet), thena2) robotic aspiration of an aspirated volume V_(a2)) of said solutionof the first polyelectrolyte PE¹, wherein the aspirated volume V_(a2))is as such that:

V _(a2)) =V _(a1)) −V _(wet).

This second alternative thus requires two additional steps beforecarrying out the n sequences. It is however interesting because itallows a homogeneous distribution of the solution on the bottom surface.Accordingly, this alternative is interesting when the volume V_(wet) isclose to V_(wet) ^(min), because low volumes of solution are moredifficult to distribute all over the surface of the bottom surface.

Usually, to ensure that the aspirated volume V_(a2)) is equal to V_(a1))minus V_(wet), the end of the tip of the robot which is used to aspiratethe solution is immersed into the solution. Accordingly, no air isaspirated and the aspirated volume, which is entered into the robot, isV_(a2)).

In this first embodiment, particularly preferred is V_(aspPE) ¹=V_(PE) ¹and V_(aspPE) ²=V_(PE) ². In this case, a constant volume of solution(i.e. V_(wet)) is left inside each well, which ensures that the surfacealways remains covered by liquid. By doing so, local differences in thewet/dry state above the polyelectrolyte film are avoided, and morehomogeneous films are obtained. Moreover, since there is a permanentliquid film inside each well wherein the film is formed, no optimizationof the distance between the end of the tip of the robots' arm and thebottom surface is required.

Typically, in this first embodiment of the method, each rinsing step isrepeated three times (i.e. each sequence of the method comprises, inthat order, steps a), b), b2), b3), b2), b3), b2), b3), b2), b3), c),d), d2), d3), d2), d3), d2), d3), d2) and d3)). As a constant volume ofsolution (i.e. V^(wet)) is left inside each well, numerous rinsing stepsare required for an effective rinsing.

In a second embodiment, called “tilting” hereafter, the plate is tilted(FIG. 1) in order to improve liquid homogeneity during the deposit andaspiration steps.

In this second embodiment, for each sequence of the method according tothis second embodiment, at steps a), b), c), d), and, if these steps arepresent, at steps b2), b3), d2) and d3), the bottom surface is tiltedwith an inclination angle α from 5 to 40° preferably from 10 to 30°,more preferably around 20°, relative to the horizontal plane,.Preferably, the angle α is the same for all the steps a), b), c), d),and, if these steps are present, for steps b2), b3), d2) and d3). Thistilting helps removing all the liquid from the well during theaspiration steps.

Typically, to tilt the bottom surface, the plate carrier is tilted withthe inclination angle α (FIG. 1).

Preferably, during the incubation steps defined above, the bottomsurface is within the horizontal plane (i.e. the bottom surface is nottilted). Accordingly, preferably, for each sequence, when each sequencecomprises steps a′) and c′) as defined above and/or comprises steps b2′)and d2′) as defined above, then, for each sequence, at steps a′) andc′), and/or at steps b2′) and d2′), the bottom surface is within thehorizontal plane. This also helps obtaining a homogeneous film, becausethe liquid is spread homogeneously at the bottom surface of the well.

Typically, the multiwell plate is tilted to implement step a), thenmoved back to an horizontal plane to implement step a′), then tilted toimplement steps b) and b2), then moved back to an horizontal plane toimplement step b2′), then tilted to implement steps b3) and c), thenmoved back to an horizontal plane to implement step c′), then tilted toimplement steps d) and d2), then moved back to an horizontal plane toimplement step d2′), then tilted to implement step d3) (FIG. 1).

Typically, in this second embodiment of the method, each rinsing step isrepeated once (i.e. each sequence of the method comprises, in thatorder, steps a), b), b2), b3), b2), b3), c), d), d2), d3), d2) and d3)).Typically, the multiwell plate is tilted to implement step a), thenmoved back to an horizontal plane to implement step a′), then tilted toimplement steps b) and b2), then moved back to an horizontal plane toimplement step b2′), then tilted to implement steps b3) and b2), thenmoved back to an horizontal plane to implement step b2′), then tilted toimplement steps b3) and c), then moved back to an horizontal plane toimplement step c′), then tilted to implement steps d) and d2), thenmoved back to an horizontal plane to implement step d2′), then tilted toimplement step d3) and d2), then moved back to an horizontal plane toimplement step d2′), then tilted to implement step d3).

In this second embodiment of the method, V_(aspPE) ¹ can be equal toV_(PE) ¹ and V_(aspPE) ² can be equal to V_(PE) ². However, in thatcase, a better homogeneity is obtained when the distance between the endof the tip of the robots' arm and the bottom surface is lower than 0.3mm. Moreover, the inventors discovered that it is very difficult toremove all the volume of solution of polyelectrolyte which was initiallydeposited, and that the remaining solution causes heterogeneity.

In order to avoid this difficulty and to improve further the homogeneityof the film, optimizing the aspiration of the volume in each well bytilting is preferably combined with aspiration of an additional volume,which enables to efficiently suck all the liquid from the well byaspirating virtually an excess volume. Accordingly, the aspirated volumeV_(aspPE) ¹ is preferably higher than V_(PE) ¹, notably from 1.05 V_(PE)¹ to 1.20×V_(PE) ¹ and the aspirated volume V_(aspPE) ² is preferablyhigher than V_(PE) ², notably from 1.05 V_(PE) ² to 1.20×V_(PE) ². Inthis case, no optimization of the distance between the end of the tip ofthe robots' arm and the bottom surface is required.

In this second embodiment, prior to step a) of the first sequence, theat least one well on which step a) will be carried out is preferablyfree from solution of polyelectrolyte (or more generally, free of anysolution, i.e. empty). The situation is thus different than the one whenthe first embodiment of the method is carried out and wherein, prior tostep a) of the first sequence, the at least one well on which step a)will be carried out comprises a solution of the second polyelectrolytePE².

This high spatial homogeneity of the obtained polyelectrolyte multilayerfilm enables to perform cell culture assays in optimized conditions. Ofspecial importance for biomedical applications is the control of thehomogeneity of the surface which can affect biological activity. Thesefilms can contain bioactive molecules and trigger controlled cellularadhesion and differentiation.

Moreover, building films directly at the bottom of multiple-well cellculture plates is of particularly interest, since this enables tocombine in situ film characterization methods and high throughputcellular assays.

Notably, absorbance, fluorescence or even luminescence measurements canbe done in routine using microplate readers.

In addition, provided that the culture plate is carefully chosen, it isalso possible to simultaneously perform optical imaging at variousresolutions, provided that the bottom of the plate is opticallytransparent and sufficiently thin to be compatible with high resolutionobjectives (×63 or above). The 96-well plate format seems especiallysuited for high throughput screening of surface coatings that containexpensive molecules, such as drugs, peptides or growth factors, since itenables to use low volumes of bioactive molecules, a low number ofcells, and to perform in situ imaging of biomolecules and cells. The96-well plate format is also particularly advantageous since it can becombined with high throughput optical and spectroscopic analysis.Notably, a 96 well plate with a thin bottom—around 180 μm—can beanalyzed using a fluorescence/absorbance microplate readers as well asimaged under a fluorescence microscope at high resolution (63× objectiveor higher).

Bioactivity of the films can be achieved by their functionalization byinserting peptides associated to polyelectrolytes or through theembedding of proteins.

Accordingly, the method can comprise, after the n sequences, a step e′)of robotic deposit of a volume V_(PE) ³ of a solution of a thirdpolyelectrolyte PE³ on said bottom surface, wherein:

-   -   polyelectrolyte PE³ is linked to at least a peptide, and    -   the third polyelectrolyte PE³ is a cationic polymer comprising        amino groups when PE² is an anionic polymer, or PE³ is an        anionic polymer when PE² is a cationic polymer comprising amino        groups.

Preferably, the peptide is RGD. Most preferably, the polyelectrolyte PE³is PGA-RGD.

Besides, in the embodiment wherein the anionic polymer comprisescarboxylic groups, the method can comprise, after the n sequences, thefollowing steps:

-   e) reacting said amino and carboxylic groups in the presence of a    coupling agent, so as to form amide bonds and to cross-link the    polyelectrolyte multilayer film, then-   f) treating said cross-linked polyelectrolyte multilayer film with a    protein containing solution, preferably with a growth factor type    protein containing solution, so as to incorporate said protein on    and inside said cross-linked polyelectrolyte multilayer film.

The coupling agent enables the formation of amide bonds (or derivativesthereof) between the carboxylic and amino groups of the polyelectrolytemultilayer. The coupling agent can act as a catalyst, which can beremoved thereafter, or as a reactant, which creates a spacer (or a link)between the formed amide bonds. For example, the coupling agent is acarbodiimide compound, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), optionally in the presence of N-hydroxysuccinimidecompounds, such as N-hydroxysulfo succinimide, more preferablyN-hydroxysulfo succinimide para-nitrophenol, or dimethylaminopyridine.

Step e) is preferably performed in a water soluble solution, morepreferably in an aqueous solution, for example in a salt free solutionor in an aqueous solution containing salts, such KCl, NaCl, or any kindof buffer such as Mes, Tris, Hepes, or phosphate buffers. Step e) ispreferably carried out at a pH ranging from 4 to 6.

The degree of crosslinking can also be controlled by varying theconcentration of the coupling agent in the solution.

Step f) is preferably carried out at a pH ranging from 2 to 9, morepreferably from 2 to 5.5.

The protein containing solution is preferably a buffer, such as Mesbuffer, preferably a solution with a low ionic strength, more preferablya buffer without any salt, such as HCl (for instance HCl at 1 mM).

Among the growth factor proteins that may be used, can be cited inparticular those that are useful for therapeutic applications and/or forcell biology applications. As examples of growth factors that may beincorporated in films according to the invention are all bonemorphogenetic proteins (BMPs), epidermal growth factors (EGF),erythropoietin (EPO), all fibroblast growth factors (FGF),granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), growth differentiation factor 5 and9 (GDF5, GDF9), hepatocyte growth factor (HGF), hepatoma derived growthfactor (HDGF), insulin-like growth factor (IGF), myostatin (GDF-8),nerve growth factor (NGF) and other neurotrophins, platelet-derivedgrowth factor (PDGF), thrombopoietin (TPO), transforming growth factors,stromal cell-derived factor-1 (SDF-1) and vascular endothelial growthfactor (VEGF), brain derived neuronal growth factor (BDNF).

Among the growth factor proteins, the transforming growth factor (TGF)family is a category of choice as this family plays an essential rolee.g. in bone formation through the regulation of osteoprogenitor andosteoblast proliferation and differentiation. Bone morphogenetic protein2 (BMP-2), a member of the TGF family, stimulates in particular thedifferentiation of myoblast cells toward an osteoblastic lineage; andits recombinant human form rhBMP-2 is more effective when deliveredassociated with a biological material such as a polymer.

The protein used at step f) can be a growth factor type protein, andpreferably a transforming growth factor. More particularly, the growthfactor is the bone morphogenetic protein 2 (BMP2) or the bonemorphogenetic protein 7 (BMP-7), the stromal cell-derived factor-1(SDF-I), or chimera 1 or chimera 2. The growth factor can be prepared byvarious methods, including biological or chemical methods. Therecombinant form is a particular embodiment. The chemical methodimplements generally automated peptide synthesizer. For instance, wildtype SDF-I can be synthesized by the Merrifield solid-phase method on afully automated peptide synthesizer using fluorenylmethyloxycarbonyl(Fmoc) chemistry.

The method can comprise, between steps e) and f) or after step f), astep g) of drying the polyelectrolyte multilayer film. This step isadvantageous to store the multiwell plate, and to transport it. Thepolyelectrolyte multilayer film can advantageously be rehydrated justbefore use.

According to a second object, the invention concerns the multiwell plateobtainable according to the method described above. This multiwell platecomprises at least one well, the bottom surface of which is coated bythe polyelectrolyte multilayer film.

This multiwell plate is characterized in that said polyelectrolytemultilayer film has a high spatial homogeneity. This spatial homogeneitycan easily be assessed, for example by the procedures and parametersdefined in the examples hereafter (notably SD and CV).

The protein loaded crosslinked polyelectrolyte multilayer films obtainedat the end of step f) are particularly useful when they are in contactwith various cell types, such as myoblast and osteoblast precursors, thecells can adhere, proliferate and optionally differentiate in a veryefficient manner. Advantageously, the cell adhesion and spreading on theobtained polyelectrolyte multilayer film is homogeneous. Cellsdistribute homogeneously on the entire surface of the well and only onepopulation of well spread cells is observed. On the contrary, when thepolyelectrolyte multilayer film is heterogeneous, the cell adhesion andspreading differs depending on the spatial position inside the well:typically, the cells are numerous and well spread at the center of thewell, but there are both less cells and less spread cells toward theborder of the well. Thus, a heterogeneous film leads to several distinctcell populations within the same well and leads to a spatialheterogeneity in cell adhesion and spreading. A homogeneouspolyelectrolyte multilayer film as obtained by the method according tothe invention is thus interesting for cell culture. Accordingly,according to a third object, the invention concerns the use of themultiwell plate for cell culture.

The following figures and examples illustrate the invention, but shouldnot be regarded as limiting the scope of the application.

FIG. 1. Schematic of the layer-by-layer deposit at high throughput inmultiple well cell culture plates. Working principle of process when thetilting embodiment is operated. The plate is tilted during all thedeposit and aspiration steps in all four major deposit and rinsingsteps: polycation and its rinsing, polyanion and its rinsing.

FIG. 2: Illustration of a multiwell plate and definition of the (X,Y, Z)coordinates, the (X,Y) coordinates of each well center being known forcommercially available cell culture plates; Z0, the initial position ofthe tip during the dispense of solutions, needs to be defined by theuser.

FIG. 3: Definition of the 4 pole positions (N, W, E, S) and centerposition (C) that are selected to assess the film thickness homogeneityinside each well. The imaging process of each of the 5 positions in eachindividual well was automatized using a custom-made macro using theconfocal microscope software.

FIG. 4: Fluorescence intensity acquired via high resolution imaging infunction of the distance Z from the bottom surface of the well. The filmthickness h=Z2−Z1 was measured using a custom-made macro using Image J.

FIG. 5: Film thickness measured at the different pole positions (N, W,C, E, S) for each well in the case of hand-made films using amultichannel pipette (comparative example). The mean+Standard deviation(mean±SD) of hN, hW, hC, hE, and hS, respectively (for m=48 wells) areplotted for each position.

FIG. 6: CV(%) measured at the different pole positions (N, W, C, E, S)for each well in the case of hand-made films using a multichannelpipette (comparative example).

FIG. 7: Box plot of the mean thickness per well (hWELL) over mindependent wells in the case of hand-made films using a multichannelpipette (comparative example).

FIG. 8: CV of hWELL (with m=48 wells) in the case of hand-made filmsusing a multichannel pipette (comparative example).

For FIGS. 5 to 8, data ware pooled for two independent experiments, eachwith 24 wells per multiwell plate (m=48 wells in total).

FIG. 9: Film thickness (μm)±SD measured at the different pole positions(N, W, C, E, S) for each well in the case of robot-made films for thecondition NT_0% of example 1.

FIG. 10: CV(%) measured at the different pole positions (N, W, C, E, S)for each well in the case of robot-made films for the condition NT_0% ofexample 1.

FIG. 11: Film thickness (μm)±SD measured at the different pole positions(N, W, C, E, S) for each well in the case of robot-made films for thecondition NT_10% of example 1.

FIG. 12: CV(%) measured at the different pole positions (N, W, C, E, S)for each well in the case of robot-made films for the condition NT_10%of example 1.

FIG. 13: Film thickness (μm)±SD measured at the different pole positions(N, W, C, E, S) for each well in the case of robot-made films for thecondition T_0% of example 1.

FIG. 14: CV(%) measured at the different pole positions (N, W, C, E, S)for each well in the case of robot-made films for the condition T_0% ofexample 1.

FIG. 15: Film thickness (μm)±SD measured at the different pole positions(N, W, C, E, S) for each well in the case of robot-made films for thecondition T_10% of example 1.

FIG. 16: CV(%) measured at the different pole positions (N, W, C, E, S)for each well in the case of robot-made films for the condition T_10% ofexample 1.

FIG. 17: Film thickness (μm)±SD measured at the different pole positions(N, W, C, E, S) for each well in the case of robot-made films for thecondition surface wet of example 1.

FIG. 18: CV(%) measured at the different pole positions (N, W, C, E, S)for each well in the case of robot-made films for the condition surfacewet of example 1.

For FIGS. 9 to 18, data are mean±SD and CV for 9 wells in total percondition, from two independent experiments.

FIG. 19: Mean thickness per well (hWELL) for (PLUHA) 12 film made in96-well plates using the robotic arm. Mean thickness per well wascalculated, for each individual microwell, as the mean of the 5positions. All mean thickness measurements were plotted as box plot foreach of the 5 experimental conditions. Data are: from 33 microwells(from 3 independent experiments) for NT conditions; for 81 wells (from 4independent experiments) for T conditions, and from 12 wells for SurfaceWet conditions (Example 1) Data representation: Data are represented asbox plots showing 1st quartile, median, 3rd quartile, the limits being10 and 90% and the extreme values 5 and 95%, respectively

FIG. 20: CV for (PLUHA) 12 film made in 96-well plates using the roboticarm corresponding to the Mean thickness per well illustrated in FIG. 19(example 1).

FIGS. 21 to 25: For each experimental condition (FIG. 21: NT_0%-FIG. 22:NT_10%-FIG. 23: T_0%-FIG. 24: T_10%-FIG. 25: surface wet), 3representative curves of the fluorescence intensities (acquired usingthe tile scan option of the confocal microscope) of all pixels in eachwell are shown (continuous, dashed and dotted black lines) (example 1).The standard deviation of their height is given in Table 2.

FIGS. 26 to 29: Thickness measurements at the five pole positions (N, W,C, E, S) and at different distances (Z-step) between the end of the tipand the bottom surface for each of the 4 experimental positions (FIG.26: NT_0%-FIG. 27: NT_10%-FIG. 28: T_0%-FIG. 29: T_10%). The end of thetip was positioned at 4 different heights above the bottom of themicroplate, from Z0=+0.1 to +1 mm by steps of +0.3 mm. (eg+0.1; +0.4;+0.7 and +1 mm above the bottom of the well (n=6 well per condition).Each box plot represents a total of 30 thickness measurements on 6independent wells at 5 positions inside each well.

FIG. 30: % of surface area covered (calculated from tile scan images ofthe microwells) by C2C12 cells for the three experimental conditions asa function of the BMP-2 initial concentration of loading. Data aremean±SD of at least 10 wells (example 3)

FIG. 31: ALP activity (a.u.) by the cells for the four experimentalconditions as a function of the BMP-2 initial concentration of loading(example 3). Bioactivity of matrix-bound BMP-2 on C2C12 cells. (PLL/HA)films built using the robotic arm were crosslinked (with EDC30),post-loaded with BMP-2 at 5, 10, 25 and 50 μg/mL were assessed for theirbioactivity. C2C12 cells plated at 5000 cells/well in growth medium werestained for ALP after 3 days of culture. (Example 3)

FIG. 32: ALP activity (a.u.) of stem cells for the T_10% condition as afunction of the BMP-7 initial concentration of loading. Bioactivity ofmatrix-bound BMP-7 on D1 stem cells. Matrix-bound BMP-7 was loaded oncrosslinked (PLUHA) 12 films (crosslinked with EDC10), which wereprepared using the robotic arm in the T_10% condition. D1 murinemesenchymal stem cells were plated in each microwell and cultured up to2 days in GM, before being switch in DM for 7 additional days. ALPactivity was quantified by enzymatic assay. 4 increasing BMP-7 loadingconcentrations from 2.5 to 50 μg/mL were tested in comparison to thefilm in the absence of BMP-7. ALP expression at day 3 is plotted as afunction of the BMP-7 loaded dose in the polyelectrolyte films. Data aremean±SD of three independent wells for each experimental condition.

FIG. 33: Film thickness of (PSS/PAH) polyelectrolyte films as a functionof n (number of layer pairs)

(PSS/PAH) films containing an increasing number of layer pairs from 10to 40 were deposited using the robotic arm with the condition T_10% on asilicon substrate using PDMS microwells. After film deposit, the PDMSwells were removed and the samples were probed by AFM. Film thicknessare measured after scratching of the films. Data are mean±SD of 25measurements (5 independent measurement per sample, 5 samples for eachexperimental condition). The linear fit of the data (Y=6,34X−7,11,R=0,974) confirms the linear growth of these films.

FIG. 34. Bioactivity of matrix-bound BMPs on the BMP responsive skeletalmyoblasts (C2C12 cells) assessed by visual observations using a scanner.ALP activity of C2C12 myoblasts cultured for 3 days on BMP-loaded films(crosslinked to EDC70) was visualized at high throughput for each singlewell by the intensity of the staining. 5 different BMPs were studied(BMP-2, BMP-4, BMP-7, BMP-9, and BV265) and 4 different BMPs loadedquantities (initial BMP concentration in solution from 2.5 to 20 μg/mL).

FIG. 35: Bioactivity of matrix-bound BMPs on the BMP responsive skeletalmyoblasts (C2C12 cells). ALP activity (a.u.) was measured as a functionof the initial concentration of BMP in solution during the loading phasein the biomimetic films. Matrix-bound BMPs were loaded on crosslinked(PLL/HA) 12 films (crosslinked with EDC70), which were prepared usingthe robotic arm in the T_10% condition. C2C12 myoblasts were plated ineach microwell and cultured for 3 days in GM. ALP activity wasquantified measuring the absorbance at 570 nm using a Tecan Infinite1000 microplate reader in a multiple reading mode (mean value of 76different positions in each single microwell). For each BMP (BMP-2,BMP-4, BMP-7, BMP-9, and BV265, 4 increasing BMPs loading concentrationsfrom 2.5 to 20 μg/mL were tested in comparison to the film in theabsence of BMPs. ALP expression at day 3 is plotted as a function of theinitial BMPs concentration in solution. Data are mean±SD of twoindependent wells for each experimental condition.

FIG. 36. Tile scan imaging of a single microwell (6.4 mm in diameter)coated with a PGA/PLL film made of 5 layer pairs. The film wasvisualized by using PLL-FITC.

FIG. 37. Number of C2C12 myoblast cells (per mm2) adhering on the on thePGA-peptide ending biomimetic films after 1 H of culture in a serum-freemedium. 4 different conditions of film crosslinking were studied (CLO,CL5, CL10, CL30) and four different PGA/PGA-RGD peptide were studied (nopeptide, ratio 2/1 ratio 1/2 and only PGA-peptide). Data are mean+SD ofthree independent well for each experimental conditions.

FIG. 38. Quantification of the myoblast cell spreading area (sameexperimental conditions as for figure XY. Cell spreading area wasautomatically quantified using a custom-made macro using Image J toncalculate the cell area covered by the cells and deduce the mean cellspreading area.

EXAMPLES Polyelectrolytes

Different types of polyelectrolytes were used for the film buildup:Poly(L-lysine) hydrobromide (PLL, Sigma, Aldrich, St Quentin Fallavier,France), Poly(allylamine hydrochloride), chitosan (CHI, FMC Biopolymers)and poly(ethylene imine) (PEI, Sigma Aldrich, France) as polycations;Hyaluronic acid (HA, Lifecore Biomedical, USA), Polystyrene sulfonate(PSS) and poly(L-glutamic acid) (PGA, both from Sigma Aldrich, France)as polyanions. PGA was grafted to a RGD containing peptide as describedin Picart et al, Adv. Funct. Mat 2005:15, 83-94)

Four different polycation/polyanion couples were selected:

-   -   the (PLL/HA) films is a model system of exponentially growing        films. For films made of 12 layer pairs, the thickness should be        around 1.5 to 2 μm, i.e. close to the resolution limit of        detection by confocal laser scanning microscopy (CLSM).    -   (PSS/PAH) films are another model system known to growth        linearly with the number of deposited layers.    -   (CHI/PGA) films were chosen as third polyelectrolyte films to        show the potentiality of the robot with other polyelectrolytes.    -   (PLL/PGA) films were chosen as fourth polyelectrolyte films to        show the potentiality of the robot to deposit other polyanions        and to do high throughput screening of cell adhesion and        spreading on films that were prepared using the robot and a film        having as final layer a mixture of PGA and PGA-RGD.

Buffers for Film Buildup

For (PLL/HA) film buildup, PLL (0.5 mg/mL), HA (1 mg/mL) and PEI (2mg/mL) were dissolved in a HEPES-NaCl buffer (20 mM Hepes at pH 7.4,0.15 M NaCl). In order to improve film adsorption to the substrates, afirst layer of PEI was deposited, followed by an HA layer. Afterwards,the cyclic deposit method of polycation (PLL) and polyanion (HA)intercalated with rinsing steps started until the desired number oflayers was reached. All rinsing steps during film buildup were performedwith 0.15 M NaCl at pH 6.5.

For (PLL/PGA) film buildup, PLL and PGA were dissolved at 1 mg/mL in theHepes-NaCl buffer.

For (PSS/PAH) film buildup, PAH and PSS were dissolved at 5 mg/mL in aTris-NaCl buffer (pH 7.4 containing X 0.15 M of NaCl⁻For CHI/PGA filmbuildup, CHI and PGA were dissolved in a 0.1 M sodium acetate buffer atpH 5 containing 0.15 M NaCl. For imaging, CHI was fluorescently labelledwith Alexa Fluor 568 (Invitrogen, Amine Reactive Probe) in accordancewith manufacturer's protocol excepting a 2 h reaction at pH 6.0. Productpurification and removal of unbound dyes was carried out using aSephadex G-25 size exclusion column (PD-10, Amersham Bioscience,Sweden). Films made of 12 layer pairs were imaged in air using the ZeissLSM 700 confocal microscope with a 10× objective.

Comparative Example: Film Deposit by Hand Using a Multiple-ChannelPipette

LbL films were built by hand in 96-well cell culture plates using amultichannel micropipette (Eppendorf Research® pro 300, Germany),typically a channel with 8 tips. The polyelectrolytes were dispensed ineach well and incubated for 8 min. Polycation and polyanion dispense wasintercalated with 2 rinsing steps of 2 min. The liquid was dispensedcarefully by tiling of the plate. It was thrown away by reversing theplate upside down. (PLL/HA) films made of 12 pairs of layers weremanually deposited in 24 wells using a 8-arm multichannel pipette.PLL-FITC was used to stain the film.

First, this procedure is tedious and requires the experimentalist to behighly focused for several hours. Besides, it is time consuming sincethe total time needed for film deposit may be very long: it isproportional to the number of deposited layer pairs n. For instance, ittakes up to two full days of work to manually prepare a film made of 24pairs of layers.

In addition, polyelectrolyte film deposition by hand reveals to behighly heterogeneous inside a single well, as can be observed on thefilm thickness analysis at the different pole positions (FIGS. 5 to 8).The coefficient of variation for the different pole positions variedbetween 10 to 23% in each specific location, and the mean thickness perwell varied of the order of 20% between independent wells (for twoindependent experiments pooled together). Without wishing to be bound bya theory, the inventors assumed that these spatial heterogeneities mayoriginate from capillary effects, which are known to be important atsuch length scale.

Example 1: Automated (PLL/HA) Film Buildup Using a Liquid HandlingMachine

A large set of in situ physico-chemical characterization and biologicalstudies was performed in the same plate: i) LbL deposit (example 1), ii)characterization of the LbL film homogeneity in situ (example 1), iii)loading of bioactive proteins and its characterization (example 3), iv)assessment of the bioactivity of the protein-loaded LbL films on cellcultures in situ in microplates: short term adhesion and ALP activitywere quantified at high throughput using optical microscopy andspectroscopy (example 3).

Automated Film Buildup Using a Liquid Handling Machine

LbL films were directly deposited in 96-wells cell culture plates(Reference 655986, Greiner bio-one, Germany) for subsequentcharacterization in situ (in liquid or air) by confocal microscopy andusing a fluorescence/absorbance microplate reader. A protocol wasdeveloped to deposit layer-by-layer films at high-throughput inmultiple-well plates using an automated liquid handling machine (TECANFreedom EVO® 100) (FIG. 1).

The film buildup with this equipment consisted in sequences ofpolycation and polyanion dispense intercalated with rinsing steps. Theprinciple consisted in using a liquid handling pipetting arm. Thisliquid handling arm pipetted the liquids in their respective reservoirsand dispensed them in selected wells of the multiple-well plate (FIG.2). The trough containing the polyelectrolytes and the rinsing solutionwere deposited on the worktable. Three through were used: one for thepolycation, one for the polyanion and one for the rinsing solutions (twoin case the rinsing solutions would be different) and a trash. Themultiple-well culture plates was/were deposited on the worktable.

We created an option for tilting the plate during dispense andaspiration steps (FIG. 1), using a commercially available tilting platecarrier: the multiple-well plate can be tilted at an angle α that isdefined by the user and can typically vary between 5 and 20 degrees, andwas 20 degrees in the examples hereafter. This condition will be namedhereafter “Tilting” (T) versus “Non Tilting” (NT) for the standardposition of the plate on the worktable (α=0 degree).

First, using a custom-made macro with the robot software, we defined thenumber of wells and specific positions where we wanted thelayer-by-layer film to be deposited.

A sequence, i.e. a pair of layers, was made of the following steps:

-   -   the liquid handling arm aspirated the polyelectrolyte from the        trough and dispense it in the selected wells (typically 50 μL)        using a tip (FIG. 2), this step being called “the dispense”,    -   incubation time in the polyelectrolyte solution of 6 min,    -   the liquid in each well was aspirated back and dispensed in the        trash. During this step, it is possible to add an additional        aspiration volume.    -   In the examples below, the defined volume is the additional        aspiration volume, defined as a % of excess volume with respect        to the volume initially deposited in that specific well (0, 5,        10, 15 or 20%, respectively corresponding to 1.00×V_(aspPE),        1.05×V_(aspPE), 1 0.15×V_(aspPE) and 1.20×V_(aspPE), as defined        above (PE being either PE¹ or PE²)); for example: if the        additional aspiration is fixed to 10%, the robot will aspirate        back 55 μL for an initial dispensed volume of 50 μL.    -   2 rinsing steps were done following the same procedure, except        that the liquid was then aspirated from the rinsing trough        (rinsing volume of 80 μL).    -   the liquid arm aspirated the oppositely-charged polyelectrolyte        (typically 50 μL) from the trough and dispensed it in the        selected wells,    -   incubation time of the oppositely-charged polyelectrolyte of 6        min.    -   the polyelectrolyte in each well was aspirated back and        dispensed in the trash.    -   x rinsing steps (typically 2) of the oppositely-charged        polyelectrolytes (typically 80 μL) were done following the same        procedure, except that the liquid was then aspirated from the        rinsing trough (rinsing volume of 80 μL).

For more precision in the pipetting, the dispenses may be achieved withthe most appropriate pipetting tip, such as 200 μL. The aspiration maybe done with a 1 mL tip.

This sequence was repeated n times to build a layer-by-layer film madeof n layer pairs.

Experimental Conditions

We compared five experimental conditions:

By controlling the tilting of the microplate (Non Tilting or Tiltingcondition with an inclination angle α of 20° relative to the horizontalplane, respectively “NT” or “T”) and the additional aspiration volume(fixed to 0% or 10%): There are four conditions in total namedhereafter:

NT_0%; NT_10%; T_0%; T_10%.

The 5th and last condition is a Non tilting/0% additional aspiration(i.e. (V_(aspPE) ¹=V_(PE) ¹ and V_(aspPE) ²=V_(PE) ²) but with apermanent excess volume during the buildup method (i.e. V^(wet)). Wename it hereafter “Surface Wet” condition.

For this condition, a volume of PE¹ polyelectrolyte solution wasdispensed inside each well at the very beginning of the experiment (fora well of a 96-well plate, this volume was set to 30 μL). This allows aconstant volume of solution to be left inside each well, in order toensure that the surface always remains covered by the liquid. By doingso, we aim to avoid local differences in the height of the liquid filmabove the polyelectrolyte film.

Pipetting Speed

The pipetting speed can be controlled by the user in the working rangeof the robotic arm. Typical pipetting speed were set to 400-800 μL/s forthe dispense step and 30-150 μL/s for the aspiration steps.

Characterization of Film Homogeneity Inside a Well (Tile Scans andTransverse Sections)

Provided that one of the film components is labelled with a fluorescentdye, it is possible to image the global film homogeneity inside a givenwell using a Tile scan option of a confocal microscope (Zeiss LSM 700,Le Peck, France) and a 10× objective. This option enables toautomatically scan the well by acquiring subsets of images.

Film Thickness “h”

We used PLL-FITC for (PLL/HA) films and (CHI-FITC) for (CHI/PGA) filmsto visualize the films. In fact, for exponentially growing films, it isknown that the last layer is able to diffuse within the whole film.Thus, the film thickness can be easily measured by measuring thethickness of the fluorescence band.

To assess the film homogeneity inside each well at high spatialresolution, we measured film thicknesses at five different positionsinside each well (North, West, Center, East and South, respectively N,W, C, E, S, FIG. 3). The center was approximately the center of thewell, as assessed by the user. The other poles were located at +2 mmfrom the center, respectively, in the X and Y directions. To measurefilm thickness at these positions (the total number of positions beingequal to the total number of wells×5), we automated the acquisition ofthe transverse sections at 0.36 μm intervals using a 63× oil objectiveand a custom-made macro with Zen software (Zeiss). Then, each thicknesswas automatically deduced from the fluorescence intensity profile (FIG.4), using a custom-made macro on Image J (NIH. Bethesda). A typicalfluorescent intensity profile starts off at the noise level (close to0), increases to a peak value as the focal plane goes deeper into thefilm, and then returns to the noise level. In brief, the maximum ofintensity (F_(MAX)) was first determined. We applied a thresholdcoefficient C so that I=C×F_(MAX). We then deduced the Z position (Z1,Z2) at which this line intercepts the fluorescence intensity profile.The film thickness h can be easily deduced: h=Z2−Z1 (in μm). Of note,this macro was validated by comparing film thicknesses measuredmanually, by atomic force microscopy and by the macro on the same image.

The film thicknesses were determined at the different positions N, W, C,E, S inside each well and the corresponding thicknesses hN, hW, hC, hEand hS were determined.

Mean Thickness Per Well “hWELL”

The mean thickness per well hWELL was calculated as the sum of all 5thickness measurements at the pole positions (N, W, E, S) and at thecenter (C), divided by 5. Thus, it was calculated using the formula:

$h_{WELL} = \frac{\left( {{hN} + {hW} + {hC} + {hE} + {hS}} \right)}{S}$

Mean Thickness of m Independent Wells “hMEAN”hMEAN was calculated as:

${hMEAN} = \frac{\sum_{1}^{m}{hWELL}}{m}$

Standard Deviation “SD” of the Mean Thickness Per Well (hWELL), for mIndependent WellsSD was calculated as:

${SD} = \sqrt{\frac{\sum_{1}^{m}\left( {{hWELLi} - {hMEAN}} \right)^{2}}{\left( {m - 1} \right)}}$

Coefficient of Variation “CV” of the Mean Thickness

The Coefficient of Variation (CV) of the mean thickness for each of the5 positions or for each well was calculated by:

${CV} = {\frac{SD}{hMEAN} \times 100}$

CV enables to compare samples independently of their absolute thicknessvalues.

Film Deposit in PDMS Microwells on Silicon Wafers for Ex SituCharacterization

Film built on silicon wafers (2″ diameter, Dow Corning, USA) were neededfor ex-situ characterization using infrared spectroscopy, profilometryand AFM microscopy.

We designed a custom-made silicon substrate with polydimethylsiloxane(PDMS, Sylgard 184 kit, Dow Corning) wells of similar size than those ofthe 96-well plates. PDMS was mixed with curing agent (10:1) during 10min and placed in a desiccator for 20 min to remove bubbles. Then, itwas introduced in a mold, degassed again during 20 min and placed in theoven at 65° C. for at least 4 h, before being carefully removed from themold and cut in rectangular pieces. Circular holes of the same diameterand position as in 96 well plates were made. Both silicon and PDMSsubstrates were UV treated (PSD-UV ozone cleaning system, NovascanTechnologies) for 10 min to increase the bonding strength between them.Thereafter, the treated faces were set in contact, pressed to adhere andintroduced in an oven at 100° C. for 1 h30 to be glued together. The(PSS/PAH) and (PLL/HA) polyelectrolyte films were then deposited usingthe robotic arm as described above.

For ex-situ film characterization an additional drying step wasmandatory.

For (PLL/HA) films, the crosslinked films stored in Hepes-Nacl bufferwere rinsed with MilliQ water and then dried.

For (PSS/PAH) films, the films were simply rinsed with MilliQ water andthen dried. Film drying was done in an incubator for 2 h at 37° C. Atthe end of the procedure, the PDMS mold was removed and the films werekept at 4° C. Before each FTIR and profilometric analysis, the filmswere placed in an incubator at 37° C. for 1 h in order to eliminate anypossible effect of humidity variation. Dry films were also characterizedby AFM.

Ex Situ Analysis Using Infrared Spectroscopy, Profilometry and AtomicForce Microscopy FTIR Analysis

Experiments were made using a Vertex 70 spectrophotometer (Bruker OpticsGmbh, Ettlingen, Germany) in the transmission mode using a sensitive MCT(Mercury-Cadmium-Telluride) detector. Prior to film analysis, abackground signal was acquired after introducing a bare siliconsubstrate in the sample compartment using the transmission accessory.Dried films built on silicon substrate were placed in a sample holderand their spectra was acquired by summing 256 interferograms. Spectraanalysis was made using OPUS Software v6.5 (Bruker, Germany), removingH₂O and CO2 contributions and correcting the baseline manually, alwayschoosing the same reference points in each spectrum. For each condition,a final spectrum is an average of 3 different spectra of the same sample(but from different wells).

Profilometry

Thickness measurements of films built on a silicon substrate wereperformed using a profilometer (Dektak XT, Bruker Corporation, USA).Five samples of each condition, corresponding to 5 different wells builtat the same time, were scratched to create a physical step and threemeasurements per sample were acquired with the software Vision 64® (v5.4, Bruker Corporation, USA). Scans of 30 s with a length of 1000 μmwere performed with a stylus of 12.5 μm in radius and with a force setto 1 mg. Thus, film thickness for each condition was an average value of15 measurements.

AFM

AFM images of the polyelectrolyte films deposited on silicon wereobtained in tapping mode by means of a DI 3100 AFM (Veeco) withNanoScope IIIa controller using silicon cantilever (OMCL-AC240TS,Olympus). The film-coated substrates were washed in water and air-driedbefore observation. Substrate topographies were imaged with 512×512pixels at a frequency of 1 Hz.

Influence of the Tilting (“T”)/Non Tilting (“NT”), of the AdditionalAspiration and of the “Surface Wet” Condition on Film Homogeneity in aSingle Well and Between Wells

Film homogeneity inside each well was assessed at the 5 pole positions(FIGS. 9 to 18). The results are provided at tables 2 and 3 below.

TABLE 2 Experimental values measured for all the parameter studied forthe 5 different experimental conditions. PARAMETER NT_0% NT_10% T_0%T_10% SW Mean ± SD of CVs of the 5 19.3 ± 9.8  20.6 ± 5.4  7.1 ± 39  5.1 ± 0.5   6.6 ± 1.6 positions CV of (hWELL) (%) 18.3  20.3 14   6.8  5.3 Global Film Homogeneity  0.742   1.004  0.172   0.177   0.494(Tile scans) Cell spreading (mm²) for 1200 ± 486 1828 ± 697 1620 ± 512BMP50 Total surface covered (%) for   7.6 ± 0.3  25.6 ± 3.9  13.0 ± 1.6BMP50 ALP bioactivity 85  83 84  83  84

In order to facilitate comparison of the five conditions, based on theexperimental values, a score was attributed for each criteria, thehigher the score, the better the parameter. A total mean score was thencalculated.

TABLE 3 Score for all the parameters studied for the 5 differentexperimental conditions. PARAMETER NT_0% NT_10% T_0% T_10% SW Mean ± SDof CVs 2 2 4 5 5 of the 5 positions CV of (hWELL) (%) 2 2 3 5 5 GlobalFilm 2 1 5 5 3.5 Homogeneity (Tile scans) Cell spreading (mm²) 1.5 5 4for BMP50 Total surface 1.5 5 3.5 covered (%) for BMP50 ALP bioactivity5 5 5 5 5 TOTAL SCORE 2.8 2.2 4.25 5 4.3

The ranking of table 3 gives the three first best conditions:T_10%>SW>T_0%. The NT_0% and NT_10% are very close and well below theothers.

Accordingly, it appears both from the absolute thickness measurements(FIGS. 9, 11, 13, 15 and 17) as well as from the CV for each pole (FIGS.10, 12, 14, 16 and 18) that:

-   -   the “surface wet” condition (first embodiment describes above),        and    -   the conditions with tilting (second embodiment describe above),    -   either with no additional aspiration (V_(aspPE) ¹=V_(PE) ¹ and        V_(aspPE) ²=V_(PE) ²),    -   or with 10% additional aspiration (V_(aspPE) ¹=1.10V_(PE) ¹ and        V_(aspPE) ²=1.10V_(PE) ²), lead to more homogeneous films.

In addition, the mean thickness per well (FIG. 19) is also much lessvariable for the experiments with tilting, especially the condition withtilting and 10% addition aspiration, and for the surface wet condition.

CV values (FIG. 20) are systematically above 15% for the “non tilting”(NT) conditions, between 10 and 15% for the T_0% condition and less than8% for both T_10% and for surface Wet.

A global view of the wells using PLL-FITC to visualize the films and thetile scan option of the confocal microscope software providedcomplimentary information on the global film homogeneity in each well.Three representative histogram of fluorescence intensities are plottedin FIGS. 21 to 25. The obtained images, as well as the histograms,clearly showed that the fluorescence distribution is more homogeneous inthe T_0% (FIG. 23) and T_10% (FIG. 24) conditions and in the Surface Wetcondition (FIG. 25) compared to the “non tilting” (NT) conditions.

Influence of the Positioning of the Pipetting Tips on the Film Thickness

Before the beginning of the experiment, the user needs to define areference position in (X,Y,Z) in order to define the initial coordinatesof the dispense and aspiration steps. The definition of (X,Y)coordinates is straightforward, knowing the coordinates of the centersof wells of a 96-well plate.

The following procedure was followed to control the Z-position of thetip at the vicinity of the plate bottom during solution dispense in thewell and aspiration from the well. Prior to the beginning of theexperiment, the tip was positioned in close vicinity to the bottom ofthe plate. The tip was first set in contact with the microplate untilthere was absolutely no movement possible for the microplate. Then, thetip was elevated in Z by one step (100 μm with the used robot). Thisposition was set as reference Z position, Z0.

We thus investigated whether and how the Z-positioning of the tipinfluences the film thickness measurement. To this end, films were builtat four different Z positions with stepwise increase of 0.3 mm from thereference position Z0 (ie 0.1 mm above the bottom of the plate) (FIGS.26 to 29).

The dispersion in film thickness increased with Z for the NT conditionsand the T_0% but the values remained almost independent of Z for theT_10% condition. Therefore, this latter condition appears to be moreflexible for the user, who does not need to be highly precise in theoptimization of Z0.

As regards the “Surface Wet” condition, since there is a permanentliquid film inside each well, there is no need to precisely adjust theinitial Z-position Z0 of the pipette tip prior to the experiments.

Example 2: Automated (PSS/PAH) or (CHI/PGA) Film Buildup Using a LiquidHandling Machine

The automated deposit method was also applied to other types ofpolyelectrolyte films. We selected a polyelectrolyte system, namely(PSS/PAH) films, that is known to grow linearly and is considered as a“model system”. As anticipated, their dry thickness was found to growthlinearly with the number of deposited layer pairs (FIG. 33) up to around250 nm for a film made of 40 pairs of layers.

We also checked for another polyelectrolyte system made of (CHI/PGA)that the polyelectrolyte film can be efficiently deposited at the bottomof the wells. The thickness of a (CHI/PGA) 12 film was 2.26±0.14 μm asmeasured from confocal microscopy imaging.

Example 3: Bioactive Proteins Loading in (PLL/HA) Films

The homogeneity of proteins that were post-loaded in the polyelectrolytefilms prepared in examples 1 and 2 was assessed.

Film Crosslinking and Loading of Bioactive Proteins in (PLL/HA) Films

Bioactive proteins were loaded in (PLL/HA) films as previously describedin Crouzier T at al., Small 2009, 5:598-608.

The films were first chemically crosslinked in a 0.15 M NaCl solution atpH 5.5 using 1-Ethyl-3-(3-Dimethylamino-propyl)Carbodiimide (EDC, finalconcentration of 10, 30 or 70 mg/mL) and N-Hydrosulfosuccinimide sodiumsalt (Sulfo-NHS, final concentration of 11 mg/mL) as catalyzer. Thefilms were incubated at 4° C. overnight, then thoroughly washed theHEPES-NaCl buffer.

The (PLL/HA) polyelectrolyte films were manually loaded with bonemorphogenetic proteins (BMP-2, BMP-7, BMP-4, BMP-9 or two BMP chimeras,namely chimera 1 and chimera 2) as bioactive proteins at acidic pH usinga multi-channel pipette, following as previously described in Crouzier Tat al., Small 2009, 5:598-608.

Cell Culture and Cell Response to the Bioactive Polyelectrolyte Films

We used BMP-2 responsive cells, C2C12 skeletal myoblasts (<25 passages,obtained from the American Type Culture Collection, ATCC), to assess thebioactivity of the polyelectrolyte films. Cells were cultured aspreviously described (Crouzier T at al., Small 2009, 5:598-608) intissue culture Petri dishes, in a 1:1 Dulbecco's Modified Eagle Medium(DMEM):Ham's F12 medium (Gibco, Invitrogen, France) supplemented with10% fetal bovine serum (FBS, PAA Laboratories, France) and 100 U/mLpenicillin G and 100 μg/mL streptomycin (Gibco, Invitrogen, France) in a37° C., 5% CO₂ incubator. Then, 15 000 cells/cm² in their medium wereseeded in each well. After 4 h and 24 h of adhesion, phase contrastimages were acquired and the samples were also fixed in 4%paraformaldehyde (Sigma Aldrich, St Quentin Fallavier, France). Thenuclei were stained using DAPI (Life technologies and the actincytoskeleton using Rhodamine-phalloidin (Sigma Aldrich).

D1 Murine Mesenchymal Stem Cell LD1) Culture

D1 cell culture was first done for 2 days in growth medium (89% aMEM(sigma M4526), 10% FBS with 1% antibiotics (penicillin streptomycin mix,15140122 Invitrogen)) followed by 7 days in differentiation medium(growth medium supplemented with 50 μg/ml L-Ascorbic acid 2-phosphateand sesquimagnesium salt hydrate (Sigma A8960) and 10 mM β-Glycerolphosphate disodium salt pentahydrate (Sigma 50020). 9375 cells wereseeded in each well. After the cell culture was stopped, ALP activitywas assessed via enzymatic assay.Human Periosteum Derived Stem Cells (hPDSC) CultureHuman periosteum derived stem cells (passage between 10 and 14) werecultured in DMEM/high glucose in the presence of 10% FBS in the presenceof 250 μM ascorbic acid 2 phosphate. They were seeded at a density a5000 cells/cm2 (˜1700 cells per well) in 200˜μL of medium. The mediumwas changed every 2-3 days and the cell culture was done for 2 weeks.

Alkaline Phosphatase (ALP) Bioactivity

After a given number of days of culture, (3 days for C2C12, 3 for D1cells and 14 for hPDSC) the growth medium was removed and the cells werefixed with 4% paraformaldehyde. They were stained for ALP activity fastblue RR salt in a 0.01% (w/v) naphthol AS-MX solution (Sigma Aldrich)according to the manufacturer's instructions. ALP enzymatic activity.

The culture medium was removed and the cells were washed with PBS andlysed by sonication over 5 s in 500 mL of 0.1% Triton-X100 in PBS. TheALP activity of these lysates was then quantified using standardprotocol and normalized to the corresponding total protein content,which was determined using a bicinchoninic acid protein assay kit(Interchim, France).

Analysis of the Homogeneity of Bioactive Proteins Loaded in thePolyelectrolyte Films

In order to assess the homogeneity of BMP-2 loading in the (PLL/HA)films, BMP-2 labelled with carboxy fluorescein (BMP-2CF) was used (5% ofthe total BMP-2 concentration) and tile scans of the wells wereperformed in the Hepes-NaCl buffer after thorough rinsing of the filmsin order to get only matrix-bound BMP-2.

The bioactivity of the BMP proteins was assessed using BMP-responsivecells. To begin, we chose to work with films crosslinked with an EDCfinal concentration of 30 mg/mL (i.e. noted EDC30 since these films areknown to be poorly adhesive for cells, unless they are presenting BMP-2in a matrix-bound manner. The more heterogeneous the film is, the moredifferences in the cell response to matrix-bound BMPs is expected.

Since matrix-bond BMP-2 on EDC30 films drastically increases celladhesion and spreading, we first assessed cell adhesion at 24 h. (FIG.30).

For these experiments, the two “extreme conditions” of NT_10% and T_10%were selected and the Surface Wet condition.

Cells appeared to be round and poorly adherent on the NT_10% conditionswhile they were more numerous and also more spread in the T_10%condition.

BMP-2 bioactivity can be quickly assessed by staining for the expressionof an early bone marker, the alkaline phosphatase (FIG. 31). Allconditions lead to a BMP-2 dose-dependent and significant ALPexpression, as can been seen after cell staining and correspondingquantifications.

High Throughput Screening of Stem Cell Adhesion and Fate

We selected the T_10% condition to further prove the versatility of thematrix-bound proteins to screen for cellular processes on stem cells athigh throughput.

We first tested whether matrix-bound BMP-7 was bioactive toward murineD1 stem cells (FIG. 32). To this end, the cells were cultured for up to9 days on the bioactive polyelectrolyte films. We found that cellsselectively adhere on the matrix-bound BMP-7 and could growth for up toat least 9 days. Of note, cells detached in the absence of matrix-boundBMP-7 and formed nodules in its presence. Their ALP expression directlydepended on the amount of matrix-bound BMP-7 and grow exponentially to aplateau value (fit in the graph of FIG. 32).

We further tested whether matrix-bound BMPs are bioactive toward murineC2C12 skeletal myoblasts (FIGS. 34 and 35).

To this end, we first verified that these BMPs could be effectivelyloaded in the biomimetic films (Table 4).

TABLE 4 Proportion and quantity of BMP loaded in the (PLL/HA) films. %incorporated Quantity (ng/cm²) SD BMP-9 86% 2325  65 BV-265 61% 1850  56BMP-2 60% 1650 115 BMP-4 65% 1810  15 BMP-7 38% 1100  84

The cells were cultured for 3 days on the bioactive polyelectrolytefilms (5 different BMP proteins and 4 different BMP loadingconcentrations). We found that cells selectively adhere on thematrix-bound BMPs and grow on this time period. Their ALP expression wasassessed at high throughput using two different methods: first, ALPstaining was visualized using a scanner and images of the wholemicroplate were taken, showing the ALP expression in each individualwell (FIG. 34). Second, ALP staining was quantified at high throughputusing a Tecan Infinite 1000 microplate reader, by quantifying theabsorbance at 570 nm using multiple-read per well mode (76 differentpositions were measured in each individual microwell and the mean valueof these 76 positions was taken). This quantification enables to plotthe ALP as a function of the initial concentration of the BMPs insolution (FIG. 35), which clearly shows a dose-dependent ALP response:the ALP intensity depends on the type of BMPs (in the orderBMP-9>BV265>BMP-2>BMP-4>BMP-7) and on the dose of BMPs (increased ALPexpression with the increased BMP concentration). The correspondingexponential fits toward a plateau value (continuous and dashes lines)are also given for BMP-2, BMP-9, BV265, BMP-4 while the fit was linearfor BMP-7 (continuous line).

Example 4: Automated (PLL/PGA) Film Buildup Using a Liquid HandlingMachine and High Throughput Screening of Cell Adhesion and Spreading

The automated deposit method was also applied to another type ofpolyelectrolyte films, namely (PGA/PLL) films that we previously studiedfor cell adhesion (Picart et al, Adv. Funct Mat 2005).

For this study, we used films made only using the T_10% condition.

The (PGA/PLL) films were made of 5 layer pairs (eg (PGA/PLL) 5 films)and were either native (eg not crosslinked, CL 0) or crosslinked todifferent extents (EDC 5, EDC10, EDC30 named hereafter as CL5, CL10,CL30). So, in total, there were 4 different films conditions. They werefinally rinsed with the Hepes-NaCl buffer using the liquid handlingmachine. On top of these films, a final layer was deposited. It isconstituted of a mixture of PGA and PGA-RGD peptide (a RGD containingpeptide grafted to the PGA) at fixed proportions. The PGA/PGA-RGD ratioused for the deposit of the final layer was varied in order to study 4different conditions for the final layer: P0(3/0); P1 (2/1); P2(1/2);P3: 0/3). C2C12 myoblast C2C12 were seeded in the 96-well microplates ata density of 3500 cells/well (around 10 500 cells/cm2).

We first verified that the biomimetic films were homogeneous inside eachwell as observed using the tile scan option of the microscope (FIG. 36,showing the whole well of about 6 mm in diameter).

Cell adhesion and spreading was next quantified after 1H of cell cultureon top of the different biomimetic films in the serum-free medium. To doso, their nucleus (stained with Hoechst) and cytoskeleton (stained withrhodamine phalloidin) were stained. Images were automatically acquiredat high throughput at 20× objective in the two channels using anautomated Zeiss fluorescence microscope. The number of adherent cellsincreases with the concentration of the RGD-peptide for theuncrosslinked films (CL 0) and films crosslinked at low extent (CL 5)but was peptide-independent for the more CL films (CL 10 and CL30) (FIG.37). Regarding the cell spreading area (FIG. 38), a clearpeptide-dependent cell spreading area was visible, with an enhancedmyoblast spreading when the quantity of peptide increased. We can thusconclude that the (PGA/PLL) biomimetic films containing a peptide-gratedlayer can be used to do cell adhesion and spreading at high throughput.

1. A multiwell plate obtainable by a method comprising n successivesequences, n being an integer from 1 to 2000, wherein each sequencecomprises the steps of: a) robotic deposit of a volume V_(PE) ¹ of asolution of a first polyelectrolyte PE¹ on the bottom surface of atleast one well of a multiwell plate, wherein the first polyelectrolytePE¹ is either a cationic polymer comprising amino groups, or an anionicpolymer, then b) robotic aspiration of an aspirated volume V_(aspPE) ¹of said solution of PE¹, wherein the aspirated volume V_(aspPE) ¹ ishigher than or equal to V_(PE) ¹, then c) robotic deposit of a volumeV_(PE) ² of a solution of a second polyelectrolyte PE² on said bottomsurface, wherein the second polyelectrolyte PE² is a cationic polymercomprising amino groups when PE¹ is an anionic polymer, or PE² is ananionic polymer when PE¹ is a cationic polymer comprising amino groups,then d) robotic aspiration of an aspirated volume V_(aspPE) ² of saidsolution of PE², wherein the aspirated volume V_(aspPE) ² is higher thanor equal to V_(PE) ².
 2. The multiwell plate according to claim 1,wherein, for each sequence of the method, at steps a), b), c), d), thebottom surface is tilted with an inclination angle α from 5 to 40°relative to the horizontal plane.
 3. The multiwell plate according toclaim 1, wherein the anionic polymer is selected from the groupconsisting of poly(acrylic) acid, poly(methacrylic) acid, poly(glutamic)acid, polyuronic acid, glycosaminoglycans, poly(aspartic acid) andPolystyrene sulfonate, any combination of the polyamino-acids (in the Dand/or L forms), and mixtures thereof.
 4. The multiwell plate accordingto claim 1, wherein the cationic polymer comprising amino group isselected from the group consisting of poly(lysine),poly(diallydimethylammonium chloride), poly(allylamine),poly(ethylene)imine, chitosan, polyarginine, Poly(ornithine),polyhistidine, poly(mannosamine), polyallylamine hydrochloride, anycombination of the polyamino acids (in the D and/or L forms), andmixtures thereof.
 5. The multiwell plate according to claim 1, whereinthe polyelectrolyte multilayer film is a poly(L-lysine)/hyaluronansodium film, a polystyrene sulfonate/polyallylamine hydrochloride film,a poly(L-lysine)/poly(L-glutamic acid) film or achitosan/poly(L-glutamic acid) film.
 6. The multiwell plate according toclaim 1, wherein the anionic polymer comprises carboxylic groups, andwherein the method comprises, after the n sequences, the followingsteps: e) reacting said amino and carboxylic groups in the presence of acoupling agent, so as to form amide bonds and to cross-link thepolyelectrolyte multilayer film, then f) treating said cross-linkedpolyelectrolyte multilayer film with a protein containing solution, soas to incorporate said protein on and inside said cross-linkedpolyelectrolyte multilayer film.
 7. The multiwell plate according toclaim 1, wherein the method comprises, after the n sequences, a step e′)of robotic deposit of a volume V_(PE) ³ of a solution of a thirdpolyelectrolyte PE³ on said bottom surface, wherein: polyelectrolyte PE³is linked to at least a peptide, and the third polyelectrolyte PE³ is acationic polymer comprising amino groups when PE² is an anionic polymer,or PE³ is an anionic polymer when PE² is a cationic polymer comprisingamino groups.
 8. A multiwell plate comprising wells, wherein the bottomsurface of m wells is coated by a polyelectrolyte multilayer film,wherein m is an integer from 1 to the number of wells of the multiwellplate, the polyelectrolyte multilayer film comprising n layer pairs, nbeing an integer from 1 to 2000 and each layer pair comprising a layerof a first polyelectrolyte PE¹ and a layer of a second polyelectrolytePE² of opposite charge, wherein the first polyelectrolyte PE¹ is eithera cationic polymer comprising amino groups, or an anionic polymer, thesecond polyelectrolyte PE² is a cationic polymer comprising amino groupswhen PE¹ is an anionic polymer, or PE² is an anionic polymer when PE¹ isa cationic polymer comprising amino groups, the polyelectrolytemultilayer film presenting a coefficient of variation CV of its meanthickness less than or equal to 20.3%, wherein${CV} = {\frac{SD}{hMEAN} \times 100}$ SD being the standard deviation${SD} = \sqrt{\frac{\sum_{1}^{m}\left( {{hWELLi} - {hMEAN}} \right)^{2}}{\left( {m - 1} \right)}}$${hMEAN} = \frac{\sum_{1}^{m}{hWELL}}{m}$$h_{WELL} = \frac{\left( {{hN} + {hW} + {hC} + {hE} + {hS}} \right)}{S}$hN, hW, hC, hE and hS being film thicknesses determined at the positionsN, W, C, E, S inside each well as shown in FIG.
 3. 9. The multiwellplate according to claim 8, wherein the anionic polymer is selected fromthe group consisting of poly(acrylic) acid, poly(methacrylic) acid,poly(glutamic) acid, polyuronic acid, glycosaminoglycans, poly(asparticacid) and Polystyrene sulfonate, any combination of the polyamino-acids(in the D and/or L forms), and mixtures thereof.
 10. The multiwell plateaccording to claim 8, wherein the cationic polymer comprising aminogroup is selected from the group consisting of poly(lysine),poly(diallydimethylammonium chloride), poly(allylamine),poly(ethylene)imine, chitosan, polyarginine, Poly(ornithine),polyhistidine, poly(mannosamine), polyallylamine hydrochloride, anycombination of the polyamino acids (in the D and/or L forms), andmixtures thereof.
 11. The multiwell plate according to claim 8, whereinthe polyelectrolyte multilayer film is a poly(L-lysine)/hyaluronansodium film, a polystyrene sulfonate/polyallylamine hydrochloride film,a poly(L-lysine)/poly(L-glutamic acid) film or achitosan/poly(L-glutamic acid) film.
 12. The multiwell plate accordingto claim 8, further comprising a layer of a third polyelectrolyte PE³deposited on the top of the polyelectrolyte multilayer film, wherein thethird polyelectrolyte PE³ is linked to at least a peptide, and the thirdpolyelectrolyte PE³ is a cationic polymer comprising amino groups whenthe second polyelectrolyte PE² is an anionic polymer, or the thirdpolyelectrolyte PE³ is an anionic polymer when the secondpolyelectrolyte PE² is a cationic polymer comprising amino groups. 13.The multiwell plate according to claim 8, wherein the anionic polymercomprises carboxylic groups, and the polyelectrolyte multilayer film iscross-linked via amide bonds or derivatives thereof formed from thecarboxylic groups and the amino groups of the polyelectrolyte multilayerfilm.
 14. The multiwell plate according to claim 13, wherein a proteinis incorporated on and inside the cross-linked polyelectrolytemultilayer film.
 15. A multiwell plate comprising wells, wherein thebottom surface of m wells is coated by a polyelectrolyte multilayerfilm, wherein m is an integer from 1 to the number of wells of themultiwell plate, the polyelectrolyte multilayer film comprising n layerpairs, n being an integer from 1 to 2000 and each layer pair comprisinga layer of a first polyelectrolyte PE¹ and a layer of a secondpolyelectrolyte PE² of opposite charge, wherein the firstpolyelectrolyte PE¹ is either a cationic polymer comprising aminogroups, or an anionic polymer, the second polyelectrolyte PE² is acationic polymer comprising amino groups when PE¹ is an anionic polymer,or PE² is an anionic polymer when PE¹ is a cationic polymer comprisingamino groups, the polyelectrolyte multilayer film presenting a standarddeviation SD less than or equal to 3.9%, wherein${SD} = \sqrt{\frac{\sum_{1}^{m}\left( {{hWELLi} - {hMEAN}} \right)^{2}}{\left( {m - 1} \right)}}$${hMEAN} = \frac{\sum_{1}^{m}{hWELL}}{m}$$h_{WELL} = \frac{\left( {{hN} + {hW} + {hC} + {hE} + {hS}} \right)}{S}$hN, hW, hC, hE and hS being film thicknesses determined at the positionsN, W, C, E, S inside each well as shown in FIG.
 3. 16. The multiwellplate according to claim 15, wherein the anionic polymer is selectedfrom the group consisting of poly(acrylic) acid, poly(methacrylic) acid,poly(glutamic) acid, polyuronic acid, glycosaminoglycans, poly(asparticacid) and Polystyrene sulfonate, any combination of the polyamino-acids(in the D and/or L forms), and mixtures thereof.
 17. The multiwell plateaccording to claim 15, wherein the cationic polymer comprising aminogroup is selected from the group consisting of poly(lysine),poly(diallydimethylammonium chloride), poly(allylamine),poly(ethylene)imine, chitosan, polyarginine, Poly(ornithine),polyhistidine, poly(mannosamine), polyallylamine hydrochloride, anycombination of the polyamino acids (in the D and/or L forms), andmixtures thereof.
 18. The multiwell plate according to claim 15, furthercomprising a layer of a third polyelectrolyte PE³ deposited on the topof the polyelectrolyte multilayer film, wherein the thirdpolyelectrolyte PE³ is linked to at least a peptide, and the thirdpolyelectrolyte PE³ is a cationic polymer comprising amino groups whenthe second polyelectrolyte PE² is an anionic polymer, or the thirdpolyelectrolyte PE³ is an anionic polymer when the secondpolyelectrolyte PE² is a cationic polymer comprising amino groups. 19.The multiwell plate according to claim 15, wherein the anionic polymercomprises carboxylic groups, and the polyelectrolyte multilayer film iscross-linked by amide bonds or derivatives thereof.
 20. The multiwellplate according to claim 15, wherein a protein is incorporated on andinside the cross-linked polyelectrolyte multilayer film.